Megalin is a glycoprotein expressed in the renal proximal tubular epithelial cells with a molecular weight of approximately 600 kDa, It is also known as the glycoprotein 330 (gp330) or low-density lipoprotein (LDL)-receptor related protein 2 (LRP2). (Non-Patent Documents 1 and 2).
Megalin serves as an endocytic receptor associated with endocytosis/resorption of a protein or the like in the proximal tubular lumen in the kidney before urinary excretion. A ligand of a resorbed protein or the like is then degraded by a lysosome in the proximal tubular epithelial cells (Non-Patent Document 3).
Clinically, a patient with diabetic nephropathy is first afflicted with diabetes, and the patient then develops microalbuminuria, which leads to continued proteinuria and then to terminal renal failure. Research that points out the significance of glomerular hyperfiltration and microalbuminuria as clinical pictures appearing in an early stage of nephropathy is featured. Specifically, it has been known that patients with type I diabetes exhibit increases in renal blood flow and glomerular filtration rate at the early phase of the disease, development of microalbuminuria was then pointed out as an early-phase change resulting in the future development of nephropathy, and the concept of early stage of nephropathy was proposed. In addition, it was discovered that the amount of albumin excreted into the urine increased to abnormal levels in this stage, although it did not lead to development of proteinuria. Thus, such symptom was designated as “microalbuminuria” (Non-Patent Document 4).
Thereafter, the clinical significance of “microalbumin” was established by Mogensen C. E., Viberti G. C. et al., and the presence of microalbuminuria is strongly correlated with the later progression of nephropathy. Thus, microalbumin is used for clinical diagnosis nowadays. It has been reported that microalbuminuria is caused by the equilibrium and failure in functions in glomerular filtration and tubular resorption (Non-Patent Documents 5, 6, 7, 8, 9, and 10).
It has also been reported that tubular albumin resorption is caused by megalin-mediated endocytosis (Non-Patent Documents 11, 12, 13, 14, 15, 16, 17, 18, and 19).
When a renal disorder advances from microalbuminuria, continued proteinuria (i.e., overt proteinuria) develops. In this stage, positive results for proteinuria are continuously found with the use of a paper test, and the disease becomes detectable with medical checkups at this stage.
According to the report of Araki S. et al., the results of a six-year follow-up study of treatment of 216 early stage of nephropathy cases demonstrated that 51% cases exhibited remission of nephropathy (i.e., improved to normal albuminuria), which occurred with higher frequency than progression (i.e., progression to overt nephropathy, 28%) (Non-Patent Document 22).
As a result of the analysis, four factors associated with remission have been exemplified: (i) a short duration following the development of microalbuminuria; (ii) use of a renin-angiotensin system inhibitor; (iii) low systolic blood pressure; and (iv) sufficient blood sugar control. Specifically, earlier diagnosis, recognition of pathological conditions, and therapeutic management of renal disorders are considered to be important to prevent advancement of diabetic nephropathy. Basic treatment methods for diabetic nephropathy are (1) blood sugar control, (2) blood pressure control, (3) suppression of the renin-angiotensin system, (4) lipid control, (5) alimentary therapy (restriction of salt and protein intake), and (6) improvement in lifestyle habits, such as smoking abstinence. In order to inhibit the development and progression of nephropathy, precise and active treatment based on evidence attained by clinical studies is necessary. Regarding blood sugar control, evidence has been attained by DCCT, UKPDS, and the Kumamoto study, which have been important for the prevention of nephropathy progression. Regarding the importance of blood pressure control, much evidence has been accumulated, including on ACE inhibitors and ARB.
Among chronic glomerulonephritis symptoms, in contrast, glomerular mesangial cell proliferation, mesangium matrix enlargement (hyperplasia), and granular deposits (mainly IgA) in the mesangial region are observed in the case of IgA nephropathy. Diagnosis of IgA nephropathy is confirmed by renal biopsy. IgA nephropathy is often detected based on asymptomatic urinary abnormalities. Continuous microscopic hematuria is inevitable, and intermittent or continuous proteinuria and macroscopic hematuria are occasionally observed. Macroscopic hematuria often occurs with acute upper respiratory tract infection. Diagnosis of urinary abnormality requires at least three instances of urine analysis, and at least two of these instances involve microscopic visualization of urinary sediments, which are conducted in addition to general qualitative urine analysis. High serum IgA levels of 315 mg/dl or higher are observed in half of the patients. A mild asymptomatic urinary abnormality should not be depreciated. Laboratory findings on IgA nephropathy are useful for the evaluation of activity and progression of renal disorders. Only hematuria is observed at an early stage, and proteinuria develops along with the progression of disease stages. Examples of unfavorable factors include high blood pressure, continuation of mild- to high-proteinuria, and renal dysfunction observed at the first medical examination. Thus, comprehensive evaluation of various laboratory findings and adequate treatment in accordance with activity of glomerulonephritis and renal disorder progression are necessary. Since IgA nephropathy is often detected by chance proteinuria/hematuria, differential diagnosis of hematuria is first required. Renal biopsy is then performed as a definite diagnosis. There are contraindications involved in renal biopsy, and there are many restrictions. In actual clinical settings, renal biopsy cannot be performed in many cases, and, at present, there is no solid, accurate indication of whether or not renal biopsy should be carried out. In renal biopsy for definite diagnosis, glomerulosclerosis symptoms from the focal and the segmental to the diffuse and the global (spherical) are evaluated with the use of mesangial proliferative changes as indicators in light microscopic findings, and diffuse glomerulosclerosis symptoms are evaluated with the use of granular IgA deposition mainly in the mesangial region as an indicator for the fluorescence antibody method or enzyme antibody method (i.e., IgA is dominant to other immunoglobulins). Evaluation is made with the deposition of electron-dense substances within the mesangial matrix (centered on a paramesangial region, in particular) as an indicator for electron microscopic findings. Prognosis based on renal biopsy is determined based on histological findings from light microscope images of renal biopsy samples, and patients are divided into the following four groups.
1) Good Prognosis Group: The Group with Little Likelihood of Progression to Dialysis Therapy
Glomerular findings: Mild mesangial cell proliferation and increased matrix are observed. No glomerulosclerosis, crescent formation, or adhesion to Bowman's capsule are observed.
Tubular, interstitial, and vascular findings: No prominent changes are observed in the renal tubule, the interstitium, or the blood vessels.
2) Relatively Good Prognosis Group: The Group with Less Likelihood of Progression to Dialysis Therapy
Glomerular findings: Mild mesangial cell proliferation and increased matrix are observed. Glomerulosclerosis, crescent formation, and adhesion to Bowman's capsule are observed in less than 10% of the biopsy glomeruli.
Tubular, interstitial, and vascular findings: No prominent changes are observed in the renal tubule, the interstitium, or the blood vessels.
3) Relatively Poor Prognosis Group: The Group with Likelihood of Transition to Dialysis Therapy in 5 to 20 Years
Glomerular findings: Moderate mesangial cell proliferation and increased matrix are observed. Glomerulosclerosis, crescent formation, and adhesion to Bowman's capsule are observed in 10% to 30% of the biopsy glomeruli.
Tubular, interstitial, and vascular findings: Slight tubular atrophy, slight cellular infiltration in the interstitium except for around some sclerosed glomeruli, and mild vascular sclerosis are observed in the blood vessels.
4) Poor Prognosis Group: The Group with likelihood of Transition to Dialysis Therapy within 5 Years
Glomerular findings: Severe mesangial cell proliferation and increased matrix are observed. Glomerulosclerosis, crescent formation, and adhesion to Bowman's capsule are observed in 30% or more of the biopsy glomeruli. When the sites of sclerosis are totalled and converted to the global sclerosis, further, the rate of glomerulosclerosis is 50% or higher. Compensatory glomerular hypertrophy is occasionally observed.
Tubular, interstitial, and vascular findings: Severe tubular atrophy, interstitial cellular infiltration, and fibrosis are observed. Hyperplasia or degeneration is occasionally observed on some renal arteriolar walls.
The basis of medication for IgA nephropathy is selection of a drug suitable for the pathological conditions of each patient. Adrenal cortical steroid therapy is suitable for a case with a creatinine clearance (Ccr) of 70 ml/min or higher, urinary protein of 1 to 2 g/day, and the acute inflammation symptoms detected by the renal biopsy as major symptoms. In contrast, drug therapy involving the use of inhibitors of the renin-angiotensin system or fish oil is selected for cases mainly involving chronic lesions and exhibiting slow progression. Renal functions of patients in the poor prognosis group exhibiting moderate or severe renal dysfunction and mainly involving chronic sclerosing lesion cannot be maintained only by steroid therapy for a long period of time, and development of effective therapeutic techniques capable of improving the prognosis of renal functions has been awaited. In addition, use of anti-platelet agents, anticoagulant therapy, Kuremejin therapy, tonsillectomy therapy, or the like is occasionally employed. IgA nephropathy occurs at a young age, and 30% to 40% of patients develop terminal renal failure. Economic and social burdens are serious when a patient has to start dialysis treatment at a young age. As described above, there are no accurate and precise indicators for differential diagnosis or diagnosis enabling prognostic prediction of IgA nephropathy at present.
The number of patients with terminal renal failure who are in need of dialysis is increasing all over the world, and it is a serious issue of concern in terms of medical economy. Prediction and diagnosis of pathological conditions of renal disorders and, in particular, diabetic nephropathy or IgA nephropathy, are most critical in order to provide adequate treatment. However, accuracy of conventional diagnostic techniques is insufficient for prognostic prediction or diagnosis of the degree of disorder.
In the preceding stage of diabetic nephropathy, microalbuminuria is not observed in urine, and nephropathy cannot be detected based on current clinical findings. Minimal albuminuria is deduced to occur at the early stage of nephropathy. Even if renal functions are normal or sometimes accelerated, nodular lesions may be present in glomeruli at this stage. Thus, whether or not microalbuminuria is useful as an indicator for early diagnosis of diabetic nephropathy remains problematic. In recent years, a case of rapidly progressive renal disorder directly leading to renal failure (stage-IV diabetic nephropathy) without the overt albuminuria stage (stage-III diabetic nephropathy (the overt nephropathy stage)) was found in the groups of diabetic nephropathy patients exhibiting microalbuminuria (stage-II diabetic nephropathy: early stage of nephropathy). Thus, the possible problematic nature of the clinical significance of the use of albuminuria for prognostic prediction of a renal disease and for precise and early diagnosis of the degree of disorder (progression in pathological conditions) as an indicator has been discussed (Perkins B. A., Krolewski A. S. et al., 2007, J. Am. Soc. Nephrol. 18 (4), 1353-1361; de Boer I. H., Steffes M. W., 2007, J. Am. Soc. Nephrol. 18 (4), 1036-1037).
IgA nephropathy prognosis is histologically classified based on renal biopsy findings, and the results are used for prognostic prediction and determination of the course of treatment. However, there are some restrictions on renal biopsy, and application of renal biopsy is restricted to the following cases: i) 1.0 g or more protein in urine is observed per day; ii) although a renal disorder of unknown etiology is observed, renal atrophy is not observed via an imaging test; iii) chronic glomerulonephritis with continuous and progressive hematuria is suspected; and iv) renal functions are rapidly lowered. Meanwhile, contraindications to renal biopsy are as follows: i) renal atrophy has already been observed via the imaging test due to chronic renal dysfunctions; ii) it is difficult to arrest hemorrhage due to bleeding tendency or uncontrollable high blood pressure; iii) a patient has a polycystic kidney; and iv) the patient cannot keep quiet or follow instructions during renal biopsy or during and after testing. In actual clinical settings, renal biopsy cannot be performed in many cases, and there are no accurate and definitive indicators for the determination of whether or not renal biopsy should be performed at present.
In addition to chronic renal disorders represented by diabetic nephropathy and IgA nephropathy described above, acute kidney injury (AKI) has drawn attention and become an issue of concern. Regarding AKI, functional abnormalities in renal hemodynamics have been regarded as critical, in addition to structural abnormalities (i.e., acute tubular necrosis) in recent years.
The term “acute renal failure” refers to a condition in which renal functions are rapidly lowered, and many acute renal failure cases are characterized by lowered renal functions caused by tubular necrosis. Causes of acute renal failure include prerenal renal failure, intrinsic renal failure, and postrenal renal failure. Prerenal renal failure occurs when the kidney is exposed to ischaemia by a lowered extracellular fluid volume due to bleeding from injury, dehydration, vomition, and diarrhea, decreased effective circulating blood volume due to cardiogenic shock, and decreased renal blood flow due to dissecting aneurysm of aorta or renal arterial thrombosis. Intrinsic renal failure is a disorder directly imposed on the renal tissue, such as in glomerular diseases (e.g., acute glomerulonephritis, rapidly progressive glomerulonephritis, and polyarteritis nodosa), acute tubular necrosis (caused by the use of an aminoglycoside antibiotics, an anti-inflammatory and analgesic agent, an anti-tumor agent, or contrast medium), or acute interstitial glomerulonephritis (caused by the use of a β-lactam antibiotics, an anti-inflammatory or analgesic agent, or an anticonvulsant). In the case of postrenal renal failure, the urine flow is obstructed and urinary excretion is prevented by ureteral obstruction (ureteral calculus), vesical and urethral obstruction (prostate hypertrophy and prostate cancer), or pelvic tumors.
Many acute renal injuries require ICU controls after cardiotomy and aorta replacement surgery, and recognition of pathological conditions is required on an hourly basis after disease development. At present, improvement in the vital prognosis for acute renal failure cannot be expected without early diagnosis and immediate intervention.
At present, acute renal failure is generally diagnosed based on serum creatinine and urine levels; however, diagnosis based on these two items suffers from a problem. That is, no diagnostic standards have been established for these two items, and there have been 35 different definitions of acute renal failure. In order to solve this problem, the acute renal failure network was established as a global effort, and diagnostic standards for acute renal failure were proposed. According to these diagnostic standards, a person is diagnosed as having acute renal failure when (1) the serum creatinine level is increased by 1.5 times or more or 0.3 mg/dl or more and (2) hypouresis of 0.5 ml/kg/hour continues for 6 hours or longer. As with the case of chronic renal disease stage classification, acute renal failure stage classification is proposed in particular (e.g., RIFLE or AKIN classification).
However, diagnosis based on the two items described above still suffers from problems. The serum creatinine level is not elevated immediately when the glomerular filtration rate is lowered due to a renal disorder. The serum creatinine level occasionally continues to increase for a while even when the glomerular filtration rate is in a recovery trend. Thus, usefulness of serum creatinine level as an early marker for monitoring acute changes or a marker for monitoring of therapeutic effects or prognostic prediction cannot be said to be satisfactory. In addition, serum creatinine level is likely to be influenced by extrarenal factors, such as body weight, race, sexuality, drugs, muscle metabolism, or nutritional conditions. Since diagnosis based on urine level takes a long time, also, it is not suitable as a marker for acute renal failure, which requires recognition of pathological conditions on an hourly basis after disease development. Therefore, development of a biomarker, which enables easy measurement, is less likely to be influenced by other biological factors, and enables early detection, risk classification, and prognostic prediction of a disease is an urgent need.